Cryptococcus neoformans, an encapsulated yeast, is a major cause of life-threatening opportunistic infections in AIDS patients and other immunocompromised patients. Little is known about the genetic structure of this pathogen. The long term goals of this project are to develop improved methods for genetic mapping in C neoformans; determine the genetic basis for differences between the two varieties of C neoformans (var. neoformans and var. gatti); determine whether C neoformans isolates from AIDS patients are different from other strains; and isolate genes affecting capsule synthesis and other virulence factors. These studies will contribute to a better understanding of the pathogenesis of cryptococcal infections and the development of new therapeutic agents. The experimental design of this project is as follows. Electrophoretic karyotypes of representative strains will be determined by pulsed field gel electrophoresis and Southern hybridization with chromosome-specific probes. Individual chromosomes will then be mapped by cloning large chromosomal fragments in yeast artificial chromosomes and aligning these clones with chromosomal restriction fragments generated by rare-cutting enzymes. These maps will be used to compare clinical isolates and to locate mutations affecting virulence factors. Genes specific to each variety or serotype mill be isolated by differential hybridization screening of cDNA subtraction libraries. These genes will be used to compare C. neoformans isolates from AIDS patients with other clinical isolates. Genes required for synthesis of the polysaccharide capsule (a major virulence determinant) will be cloned by complementation of a capsular mutants and sib selection using a genomic library. Genes required for growth in physiological concentrations of CO2 will be identified by differential hybridization screening and by isolation of CO2-sensitive mutants.